CN113881621B - Embryonic stem cell culture medium and preparation method and application thereof - Google Patents

Embryonic stem cell culture medium and preparation method and application thereof Download PDF

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CN113881621B
CN113881621B CN202111155579.5A CN202111155579A CN113881621B CN 113881621 B CN113881621 B CN 113881621B CN 202111155579 A CN202111155579 A CN 202111155579A CN 113881621 B CN113881621 B CN 113881621B
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embryonic stem
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陈东煌
陈海佳
姜交华
张兆清
李学家
王悦萌
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Guangdong Guoke Cell Technology Co ltd
Bioisland Laboratory
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Abstract

The invention relates to an embryonic stem cell culture medium, a preparation method and application thereof, wherein the culture medium comprises a basic culture medium and the following additive components in amount: 1-50 mug/L sodium selenite, 100-400 mug/L ferric ethylenediamine tetraacetate, 5-100 mug/L recombinant human basic fibroblast growth factor, 1 nM-50 nM insulin primary growth factor, 1-10 mg/L reduced glutathione, 1-100 mg/L ascorbic acid, 1-20 mug/L recombinant human transforming growth factor beta 1, 0.1-5 ng/mL recombinant human regulatory protein beta-1, 1 nM-100 nM hexokinase and 0.1-10 mug M N-oxaloglycine. The embryonic stem cell culture medium is used for culturing the embryonic stem cells, so that the pluripotency of the embryonic stem cells can be effectively maintained.

Description

Embryonic stem cell culture medium and preparation method and application thereof
Technical Field
The invention relates to the technical field of cell culture, in particular to an embryonic stem cell culture medium and a preparation method and application thereof.
Background
Embryonic Stem Cells (ESCs) have unlimited self-renewal, multipotent protein expression, high telomerase activity, and the ability to differentiate toward the tricodendron. ESCs have wide application prospects in aspects of disease modeling, drug screening and the like. However, in the process of culturing embryonic stem cells in vitro, there are often problems of cell differentiation, heterogeneity, and the like. Therefore, how to maintain the pluripotency of embryonic stem cells is an urgent problem to be solved.
Disclosure of Invention
Based on the background art, the invention mainly aims to provide an embryonic stem cell culture medium and a preparation method and application thereof. The embryonic stem cell culture medium is used for culturing embryonic stem cells, and can effectively maintain the pluripotency of the embryonic stem cells.
The aim of the invention can be achieved by the following technical scheme:
a composition comprising N-oxaloglycine, recombinant human regulatory protein β -1 and hexokinase.
In one embodiment, the molar ratio of N-oxalylglycine, recombinant human regulatory protein beta-1 to hexokinase is (0.1-10): (1.333X 10) -5 ～6.667×10 -4 ）：（1×10 -3 ～100×10 -3 ）。
In one embodiment, the molar ratio of N-oxalylglycine, recombinant human regulatory protein beta-1 to hexokinase is (1-10): (1.333X 10) -4 ～6.667×10 -4 ）：（10×10 -3 ～100×10 -3 ）。
An embryonic stem cell culture medium comprising the composition, further comprising the following media components: basic culture medium, sodium selenite, ferric ethylenediamine tetraacetate, recombinant human basic fibroblast growth factor, insulin-like growth factor I, reduced glutathione, ascorbic acid and recombinant human transforming growth factor beta 1.
In one embodiment, the embryonic stem cell culture medium comprises 0.1. Mu.M to 10. Mu.M of the N-oxaloglycine, 13.33pM to 666.7pM of the recombinant human regulatory protein beta-1, and 1nM to 100nM of the hexokinase.
In one embodiment, the embryonic stem cell culture medium comprises 1. Mu.M to 10. Mu.M of the N-oxaloglycine, 133.3pM to 666.7pM of the recombinant human regulatory protein beta-1, and 10nM to 100nM of the hexokinase.
In one embodiment, the medium comprises basal medium, 1 μg/L-50 μg/L sodium selenite, 100 μM-400 μM ferric ethylenediamine tetraacetate, 5 μg/L-100 μg/L recombinant human basic fibroblast growth factor, 1 nM-50 nM insulin type I growth factor, 1 mg/L-10 mg/L reduced glutathione, 1 mg/L-100 mg/L ascorbic acid, 1 μg/L-20 μg/L recombinant human transforming growth factor β1, 13.33 pM-666.7 pM recombinant human regulatory protein β -1, 1 nM-100 nM hexokinase, and 0.1 μM-10 μ M N-oxaloglycine.
In one embodiment, the medium comprises a basal medium and the following amounts of additive components: 1-50 mug/L sodium selenite, 100-400 mug/L ferric ethylenediamine tetraacetate, 5-100 mug/L recombinant human basic fibroblast growth factor, 1 nM-50 nM insulin primary growth factor, 1-10 mg/L reduced glutathione, 1-100 mg/L ascorbic acid, 1-20 mug/L recombinant human transforming growth factor beta 1, 133.3 pM-666.7 pM recombinant human regulatory protein beta-1, 10 nM-100 nM hexokinase and 1 mug-10 mug M N-oxaloglycine.
In one embodiment, the medium comprises a basal medium and the following amounts of additive components: 20-40 mug/L sodium selenite, 250-350 mug/L ferric ethylenediamine tetraacetate, 30-50 mug/L recombinant human basic fibroblast growth factor, 5-20 nM insulin primary growth factor, 1-5 mg/L reduced glutathione, 50-70 mg/L ascorbic acid, 1-5 mug/L recombinant human transforming growth factor beta 1, 133.3-666.7 pM recombinant human regulatory protein beta-1, 10-100 nM hexokinase and 1-10 mug M N-oxaloglycine.
In one embodiment, the basal medium is a DMEM/F12 basal medium.
A method of preparing an embryonic stem cell culture medium as described above, the method comprising the steps of: mixing the medium components and the composition.
A method of culturing embryonic stem cells, the method comprising the step of inoculating embryonic stem cells into an embryonic stem cell culture medium for culturing, the embryonic stem cell culture medium being an embryonic stem cell culture medium as described above.
In one embodiment, the culturing method further comprises: fresh embryonic stem cell medium is replaced during the culturing process.
In one embodiment, the culturing method further comprises: and adopting a subculture reagent for subculture in the culture process.
In one embodiment, the subculture reagent is used for 4-6 d per culture.
In one embodiment, the embryonic stem cells are huES (H9) cells.
In one embodiment, the embryonic stem cells are embryonic stem cells cultured to passage 25 to 35.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, three components of hexokinase, N-oxaloglycine, recombinant human regulatory protein beta-1, sodium selenite, ferric ethylenediamine tetraacetate, recombinant human basic fibroblast growth factor, insulin-like growth factor I, reduced glutathione, ascorbic acid and recombinant human transforming growth factor beta 1 are matched as additive components and added into a basal medium to form an embryonic stem cell culture medium with a specific formula, and the embryonic stem cell culture medium is adopted to culture embryonic stem cells, so that the pluripotency of the embryonic stem cells can be effectively maintained. In addition, the inventor also discovers that the expansion times of the embryonic stem cells can be obviously improved by adopting the embryonic stem cell culture medium to culture the embryonic stem cells, and the survival rate of the embryonic stem cells is higher. Meanwhile, the embryonic stem cell culture medium is a serum-free culture medium, the additive components are definite, animal-derived components are avoided, and the toxic effect of serum on cells and serum-derived pollution are avoided.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a morphology of the 42 th generation huES (H9) cells;
FIG. 2 shows the fold expansion of the 42 th generation huES (H9) cells;
FIG. 3 shows cell viability of the 42 th generation huES (H9) cells;
FIG. 4 is a diagram showing Oct4 gene expression in 42 th generation huES (H9) cells;
FIG. 5 is a diagram showing the Sox2 gene expression in 42 th generation huES (H9) cells;
FIG. 6 is a diagram showing the expression of Nanog gene in huES (H9) cell at 42 th generation.
Description of the embodiments
The present invention will be described in more detail below in order to facilitate understanding of the present invention. It should be understood, however, that the invention may be embodied in many different forms and is not limited to the implementations or embodiments described herein. Rather, these embodiments or examples are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments or examples only and is not intended to be limiting of the invention. As used herein, the optional scope of the term "and/or" includes any one of the two or more related listed items, as well as any and all combinations of related listed items, including any two or more of the related listed items, or all combinations of related listed items.
In the present invention, "first aspect", "second aspect", "third aspect", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of the indicated technical features.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present invention, the numerical range is referred to, and both ends of the numerical range are included unless otherwise specified.
The percentage content referred to in the present invention refers to mass percentage for both solid-liquid mixing and solid-solid mixing and volume percentage for liquid-liquid mixing unless otherwise specified.
The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system after the component is added.
The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a predetermined temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.
In a first aspect, the invention provides a composition comprising N-oxaloglycine, recombinant human regulatory protein β -1 and hexokinase.
In one example, the molar ratio of N-oxaloglycine, recombinant human regulatory protein β -1 to hexokinase is (0.1-10): (1.333X 10) -5 ～6.667×10 -4 ）：（1×10 -3 ～100×10 -3 ）。
In one example, the molar ratio of N-oxaloglycine, recombinant human regulatory protein β -1 to hexokinase is (1-10): (1.333X 10) -4 ～6.667×10 -4 ）：（10×10 -3 ～100×10 -3 ）。
In a second aspect, the present invention provides an embryonic stem cell culture medium, in particular a serum-free embryonic stem cell culture medium, comprising the composition, further comprising the following medium components: basic culture medium, sodium selenite, ferric ethylenediamine tetraacetate, recombinant human basic fibroblast growth factor, insulin-like growth factor I, reduced glutathione, ascorbic acid and recombinant human transforming growth factor beta 1.
In one example, the embryonic stem cell culture medium comprises 0.1. Mu.M to 10. Mu.M of the N-oxaloglycine, 13.33pM to 666.7pM of the recombinant human regulatory protein beta-1, and 1nM to 100nM of the hexokinase.
In one example, the embryonic stem cell culture medium comprises 1. Mu.M to 10. Mu.M of the N-oxaloglycine, 133.3pM to 666.7pM of the recombinant human regulatory protein beta-1, and 10nM to 100nM of the hexokinase.
In one example, the embryonic stem cell culture medium comprises basal medium, 1 μg/L-50 μg/L sodium selenite, 100 μM-400 μM ferric ethylenediamine tetraacetate, 5 μg/L-100 μg/L recombinant human basic fibroblast growth factor, 1 nM-50 nM insulin type I growth factor, 1 mg/L-10 mg/L reduced glutathione, 1 mg/L-100 mg/L ascorbic acid, 1 μg/L-20 μg/L recombinant human transforming growth factor β1, 13.33 pM-666.7 pM recombinant human regulatory protein β -1, 1 nM-100 nM hexokinase, and 0.1 μM-10 μ M N-oxaloglycine.
According to the invention, three components of hexokinase, N-oxaloglycine, recombinant human regulatory protein beta-1, sodium selenite, ferric ethylenediamine tetraacetate, recombinant human basic fibroblast growth factor, insulin-like growth factor I, reduced glutathione, ascorbic acid and recombinant human transforming growth factor beta 1 are matched as additive components and added into a basal medium to form an embryonic stem cell culture medium with a specific formula, and the embryonic stem cell culture medium is adopted to culture embryonic stem cells, so that the pluripotency of the embryonic stem cells can be effectively maintained.
Cell energy metabolism is closely related to cell fate, and embryonic stem cells have unique metabolic characteristics and energy requirements, particularly high glycolysis is critical for maintenance of the pluripotency of ESCs. The embryonic stem cell culture medium comprises the following components: hexokinase, a transferase, acts against a variety of hexoses, and has the effect of maintaining glycolysis; n-oxaloglycine is an inhibitor of alpha-ketoglutarate-dependent enzyme, and can inhibit metabolism of mitochondria and improve glycolysis process; the regulatory protein, heregulin beta-1, called neuregulin-1, activates MAPK/ERK signaling pathway after intracellular binding to ErbB3/ErbB4, playing an important role in maintaining survival and growth of human embryonic stem cells.
In addition, the inventor also discovers that the expansion times of the embryonic stem cells can be obviously improved by adopting the embryonic stem cell culture medium to culture the embryonic stem cells, and the survival rate of the embryonic stem cells is higher. Meanwhile, the embryonic stem cell culture medium is a serum-free culture medium, the additive components are definite, animal-derived components are avoided, and the toxic effect of serum on cells and serum-derived pollution are avoided.
In one preferred example, the medium comprises a basal medium and the following amounts of additive components: 1-50 mug/L sodium selenite, 100-400 mug/L ferric ethylenediamine tetraacetate, 5-100 mug/L recombinant human basic fibroblast growth factor, 1 nM-50 nM insulin primary growth factor, 1-10 mg/L reduced glutathione, 1-100 mg/L ascorbic acid, 1-20 mug/L recombinant human transforming growth factor beta 1, 133.3 pM-666.7 pM recombinant human regulatory protein beta-1, 10 nM-100 nM hexokinase and 1 mug-10 mug M N-oxaloglycine.
In a further preferred example, the medium comprises a basal medium and the following amounts of additive components: 20-40 mug/L sodium selenite, 250-350 mug/L ferric ethylenediamine tetraacetate, 30-50 mug/L recombinant human basic fibroblast growth factor, 5-20 nM insulin primary growth factor, 1-5 mg/L reduced glutathione, 50-70 mg/L ascorbic acid, 1-5 mug/L recombinant human transforming growth factor beta 1, 133.3-666.7 pM recombinant human regulatory protein beta-1, 10-100 nM hexokinase and 1-10 mug M N-oxaloglycine.
It will be appreciated that the invention is not particularly limited in the nature of the basal medium, as long as it is sufficient for culturing embryonic stem cells, including but not limited to DMEM/F12 basal medium.
In a further preferred example, the medium comprises DMEM/F12 basal medium and additive components in the following amounts: 20 mu g/L-40 mu g/L sodium selenite, 250 mu M-350 mu M ethylenediamine tetraacetic acid iron, 30 mu g/L-50 mu g/L recombinant human basic fibroblast growth factor, 5 nM-20 nM insulin-like growth factor I, 1 mg/L-5 mg/L reduced glutathione, 50 mg/L-70 mg/L ascorbic acid, 1 mu g/L-5 mu g/L recombinant human transforming growth factor beta 1, 1 ng/mL-5 ng/mL recombinant human regulatory protein beta-1, 10 nM-100 nM hexokinase and 1 mu M-10 mu M N-oxaloglycine.
In a third aspect, the present invention provides a method for preparing an embryonic stem cell culture medium as described above, the method comprising the steps of: mixing the medium components and the composition.
It will be appreciated that the additive components of the present invention may be sterilized prior to mixing with the basal medium by, but not limited to, filter sterilization, e.g., by preparing a solution of the additive components and then filter sterilizing the solution using a 0.22 μm filter, and the filter sterilized solution may be stored at-20 ℃ for later use. After mixing the additive components with the basal medium, the basal medium may be stored at 4 ℃ for later use.
In a fourth aspect, the present invention provides a method of culturing embryonic stem cells, the method comprising the step of inoculating the embryonic stem cells into an embryonic stem cell culture medium for culturing, the embryonic stem cell culture medium being an embryonic stem cell culture medium as described above.
It will be appreciated that in the above culture methods, fresh culture medium for the embryonic stem cells is replaced during the culture process. For example, the embryonic stem cell culture medium may be replaced daily beginning on the next day of passage.
It will be appreciated that in the above culture method, the subculture reagent is used for subculture during the culture. For example, the subculture reagent is used for subculture for 4d to 6d (e.g., 4d, 5d, 6 d) per culture. It will be appreciated that the invention is not particularly limited to the type of subculture reagent in question, including but not limited to ReleSR.
It is to be understood that the type of embryonic stem cells in the culture method is not particularly limited, and may be huES (H9) cells, for example.
It is to be understood that in the above-described culture method, the invention is not particularly limited in terms of the number of passages of embryonic stem cells which are the object of culture, for example, embryonic stem cells which are cultured to 25 th to 35 th passages, such as 25 th passages, 27 th passages, 29 th passages, 31 th passages, 33 th passages, 35 th passages.
The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials are commercially available unless otherwise indicated. The components and reagents referred to in the following examples and comparative examples are conventional commercial products, in which DMEM/F12 basal medium is purchased from Gibco company and the other components are purchased from sigma, peprotech, gibco company. The control group used STEM CELL company TeSR-E8 medium.
TABLE 1 Material concentration conversion Table
Figure SMS_1
Examples
The embodiment provides a serum-free culture medium for embryonic stem cells and a preparation method thereof.
The serum-free culture medium for the embryonic stem cells comprises a basal culture medium and the following additive components in the amount: 30 μg/L sodium selenite, 300 μg/L ferric ethylenediamine tetraacetate, 40 μg/L recombinant human basic fibroblast growth factor, 10nM insulin primary growth factor, 2mg/L reduced glutathione, 64mg/L ascorbic acid, 2 μg/L recombinant human transforming growth factor beta 1, 1ng/mL recombinant human regulatory protein beta-1, 10nM hexokinase and 1 μ M N-oxaloglycine.
The preparation method of the embryonic stem cell serum-free medium comprises the following steps:
dissolving the above additive components according to respective dissolution characteristics, filtering with 0.22 μm filter membrane for sterilization, and preserving the additive at-20deg.C; before culturing the cells, adding the cells into a DMEM/F12 basal medium at 20 ℃ under aseptic conditions, blowing and uniformly mixing, and preserving the prepared embryonic stem cells in a serum-free medium at 4 ℃.
Example 2
The embodiment provides a serum-free culture medium for embryonic stem cells and a preparation method thereof.
The serum-free culture medium for the embryonic stem cells comprises a basal culture medium and the following additive components in the amount: 30 μg/L sodium selenite, 300 μg/L ferric ethylenediamine tetraacetate, 40 μg/L recombinant human basic fibroblast growth factor, 10nM insulin primary growth factor, 2mg/L reduced glutathione, 64mg/L ascorbic acid, 2 μg/L recombinant human transforming growth factor beta 1, 5ng/mL recombinant human regulatory protein beta-1, 10nM hexokinase and 1 μ M N-oxaloglycine.
The preparation method of the embryonic stem cell serum-free medium is described in example 1.
Example 3
The embodiment provides a serum-free culture medium for embryonic stem cells and a preparation method thereof.
The serum-free culture medium for the embryonic stem cells comprises a basal culture medium and the following additive components in the amount: 30 μg/L sodium selenite, 300 μg/L ferric ethylenediamine tetraacetate, 40 μg/L recombinant human basic fibroblast growth factor, 10nM insulin primary growth factor, 2mg/L reduced glutathione, 64mg/L ascorbic acid, 2 μg/L recombinant human transforming growth factor beta 1, 1ng/mL recombinant human regulatory protein beta-1, 100nM hexokinase and 1 μ M N-oxaloglycine.
The preparation method of the embryonic stem cell serum-free medium is described in example 1.
Example 4
The embodiment provides a serum-free culture medium for embryonic stem cells and a preparation method thereof.
The serum-free culture medium for the embryonic stem cells comprises a basal culture medium and the following additive components in the amount: 30 μg/L sodium selenite, 300 μg/L ferric ethylenediamine tetraacetate, 40 μg/L recombinant human basic fibroblast growth factor, 10nM insulin-like growth factor I, 2mg/L reduced glutathione, 64mg/L ascorbic acid, 2 μg/L recombinant human transforming growth factor beta 1, 1ng/mL recombinant human regulatory protein beta-1, 10nM hexokinase and 10 μ M N-oxaloglycine.
The preparation method of the embryonic stem cell serum-free medium is described in example 1.
Table 2, summary of the serum-free Medium formulations of embryonic Stem cells of examples 1 to 4
Figure SMS_2
Examples
The embodiment provides a serum-free culture medium for embryonic stem cells and a preparation method thereof.
The serum-free culture medium for the embryonic stem cells comprises a basal culture medium and the following additive components in the amount: 1. Mu.g/L sodium selenite, 100. Mu.M ferric ethylenediamine tetraacetate, 5. Mu.g/L recombinant human basic fibroblast growth factor, 1nM insulin-like growth factor I, 1mg/L reduced glutathione, 1mg/L ascorbic acid, 1. Mu.g/L recombinant human transforming growth factor beta 1, 1ng/mL recombinant human regulatory protein beta-1, 10nM hexokinase and 10. Mu. M N-oxaloglycine.
The preparation method of the embryonic stem cell serum-free medium is described in example 1.
Example 6
The embodiment provides a serum-free culture medium for embryonic stem cells and a preparation method thereof.
The serum-free culture medium for the embryonic stem cells comprises a basal culture medium and the following additive components in the amount: 50. Mu.g/L sodium selenite, 400. Mu.M ferric ethylenediamine tetraacetate, 100. Mu.g/L recombinant human basic fibroblast growth factor, 50nM insulin-like growth factor I, 10mg/L reduced glutathione, 100mg/L ascorbic acid, 20. Mu.g/L recombinant human transforming growth factor beta 1, 1ng/mL recombinant human regulatory protein beta-1, 10nM hexokinase and 10. Mu. M N-oxaloglycine.
The preparation method of the embryonic stem cell serum-free medium is described in example 1.
Example 7
The embodiment provides a serum-free culture medium for embryonic stem cells and a preparation method thereof.
The serum-free culture medium for the embryonic stem cells comprises a basal culture medium and the following additive components in the amount: 50 μg/L sodium selenite, 400 μg/L ferric ethylenediamine tetraacetate, 100 μg/L recombinant human basic fibroblast growth factor, 50nM insulin-like growth factor I, 10mg/L reduced glutathione, 100mg/L ascorbic acid, 20 μg/L recombinant human transforming growth factor beta 1, 5ng/mL recombinant human regulatory protein beta-1, 100nM hexokinase and 10 μ M N-oxaloglycine.
The preparation method of the embryonic stem cell serum-free medium is described in example 1.
Table 3, serum-free medium formulations of embryonic stem cells of examples 5-7
Figure SMS_3
This comparative example is a comparative example of example 1, and the main differences with respect to example 1 include: the additive component is free of recombinant human regulatory protein (Heregulin) beta-1.
The other components and amounts were the same as in example 1. Likewise, the preparation is also referred to in example 1.
Comparative example 2
This comparative example is a comparative example of example 1, and the main differences with respect to example 1 include: the additive component is free of hexokinase.
The other components and amounts were the same as in example 1. Likewise, the preparation is also referred to in example 1.
Comparative example 3
This comparative example is a comparative example of example 1, and the main differences with respect to example 1 include: the additive component is free of N-oxalylglycine.
The other components and amounts were the same as in example 1. Likewise, the preparation is also referred to in example 1.
Comparative example 4
This comparative example is a comparative example of example 1, and the main differences with respect to example 1 include: the additive component does not contain two components of recombinant human Heregulin beta-1 and hexokinase.
The other components and amounts were the same as in example 1. Likewise, the preparation is also referred to in example 1.
Comparative example 5
This comparative example is a comparative example of example 1, and the main differences with respect to example 1 include: the additive component does not contain two components of hexokinase and N-oxaloglycine.
The other components and amounts were the same as in example 1. Likewise, the preparation is also referred to in example 1.
Comparative example 6
This comparative example is a comparative example of example 1, and the main differences with respect to example 1 include: the additive component does not contain two components of recombinant human Heregulin beta-1 and N-oxalylglycine.
The other components and amounts were the same as in example 1. Likewise, the preparation is also referred to in example 1.
Comparative example 7
This comparative example is a comparative example of example 1, and the main differences with respect to example 1 include: the additive component does not contain three components of recombinant human Heregulin beta-1, hexokinase and N-oxalylglycine.
The other components and amounts were the same as in example 1. Likewise, the preparation is also referred to in example 1.
Comparative example 8
This comparative example is a comparative example of example 1, and the main differences with respect to example 1 include: the amounts of the three components of recombinant human Heregulin beta-1, hexokinase and N-oxaloglycine are divided into 6ng/mL recombinant human regulatory protein beta-1, 110nM hexokinase and 12 mu M N-oxaloglycine.
The other components and amounts were the same as in example 1. Likewise, the preparation is also referred to in example 1.
Application example 1, morphology observation of ESCs
The 32 nd generation huES (H9) cells were cultured according to 1X 10 5 cell/cm 2 Inoculating the culture medium to a 6-well plate coated with Vitronectin, respectively adding the serum-free culture medium of each group of embryonic stem cells for culture, changing the liquid every day from 2 nd day, adopting the ReleaseSR to perform continuous subculture every 4-6 days, culturing until 42 th generation, observing the morphology of ESCs in each 42 th generation under an inverted microscope, and collecting images.
As shown in FIG. 1, only the cell states of examples 1 to 7 and the control group (TeSR-E8 medium) were good, no significantly differentiated cells were found, the cloning edge was smooth, the cell states of the other control groups were different degrees from each other, significantly differentiated cells were found, the differentiated cells were in the shape of a shuttle or polygon, and the nuclear-cytoplasmic ratio was decreased.
Application example 2, ESCs amplification and Activity detection
huES (H9) cells cultured to passage 42 were cultured according to 2X 10 4 cell/cm 2 Is inoculated into a Vitonictin coated 24-well plate, 3 multiple wells per group. The culture medium of each group is added for culture, the liquid is changed every day from the 2 nd day, the culture is carried out until the 7 th day, and the cells are collected every day to calculate the expansion times and the cell activity rate of each group. The results are shown in Table 4, FIG. 2 and FIG. 3.
FIG. 2 shows that the amplification factors of examples 1 to 7 and the control group are high, the amplification factor is more than 40 times after 7 days of culture, and the amplification factor of example 1 is as high as 50 times. The other comparative examples had a cell proliferation phenomenon, but the cell cycle was prolonged and the amplification rate was affected due to the presence of differentiated cells, and the total amplification factor was lower than in examples 1 to 7 and the comparative examples.
From the cell viability results of FIG. 3, it can also be seen that the viability of examples 1 to 7 was better maintained (all greater than 94%), indicating that the serum-free culture medium for embryonic stem cells according to the present invention has a stronger ability to promote cell proliferation and maintain cell viability.
TABLE 4 amplification factors and Activity detection of ESCs under culture in various groups of Medium
Figure SMS_4
The huES (H9) cells cultured to the 42 th generation were cultured according to 1X 10 5 cell/cm 2 Is inoculated in 6-well plates coated with Vitronin, and each group of culture medium is added for culture. The liquid is changed every day from the 2 nd day, and the culture is carried out until the 5 th day. And (3) respectively extracting total RNA of each group of huES (H9) cells after 0.25% pancreatin digestion, reversely transcribing the total RNA into cDNA, detecting the expression level of each group of ESCs multipotent genes Oct4, sox2 and Nanog by adopting fluorescent quantitative PCR by taking the cDNA as a template, wherein the qPCR primer sequences are shown in table 5.
As shown in fig. 4 to 6, the expression levels of ESCs pluripotency genes Oct4, sox2, nanog of examples 1 to 7 were improved to different degrees as compared with the control group, and comparative examples 1 to 8 also caused a significant decrease in the expression levels of pluripotency genes due to cell differentiation. The serum-free culture medium additive for the embryonic stem cells can better maintain the pluripotency of ESCs.
TABLE 5 hESCs pluripotency gene qPCR primer sequences
Figure SMS_5
In summary, the invention uses three components of hexokinase, N-oxaloglycine, recombinant human regulatory protein beta-1, sodium selenite, ferric ethylenediamine tetraacetate, recombinant human basic fibroblast growth factor, insulin-like growth factor I, reduced glutathione, ascorbic acid and recombinant human transforming growth factor beta 1 as additive components to be added into basic culture, thus forming embryonic stem cell culture medium with specific formula, and the embryonic stem cell culture medium is adopted to culture embryonic stem cells, so that the pluripotency of the embryonic stem cells can be effectively maintained. In addition, the inventor also discovers that the expansion times of the embryonic stem cells can be obviously improved by adopting the embryonic stem cell culture medium to culture the embryonic stem cells, and the survival rate of the embryonic stem cells is higher. Meanwhile, the embryonic stem cell culture medium is a serum-free culture medium, the additive components are definite, animal-derived components are avoided, and the toxic effect of serum on cells and serum-derived pollution are avoided.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. It should be understood that, based on the technical solutions provided by the present invention, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.
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Claims (6)

1. An embryonic stem cell culture medium is characterized by comprising a basic culture medium, 1-50 mug/L sodium selenite, 100-400 mug/L ferric ethylenediamine tetraacetate, 5-100 mug/L recombinant human basic fibroblast growth factor, 1 nM-50 nM insulin type I growth factor, 1-10 mg/L reduced glutathione, 1-100 mg/L ascorbic acid, 1-20 mug/L recombinant human transforming growth factor beta 1, 1-10 mug/L M N-oxalylglycine, 133.3 pM-666.7 pM recombinant human regulatory protein beta-1 and 10 nM-100 nM of hexokinase;
the basal medium is DMEM/F12 basal medium.
2. The embryonic stem cell culture medium of claim 1, comprising basal medium, 30 μg/L sodium selenite, 300 μΜ ferric ethylenediamine tetraacetate, 40 μg/L recombinant human basic fibroblast growth factor, 10nM insulin-like growth factor No. 1, 2mg/L reduced glutathione, 64mg/L ascorbic acid, 2 μg/L recombinant human transforming growth factor β1, 1 μΜ -10 μ M N-oxaloglycine, 133.3 pM-666.7 pM recombinant human regulatory protein β -1 and 10 nM-100 nM of the hexokinase.
3. A method of preparing an embryonic stem cell culture medium according to any one of claims 1 to 2, comprising the steps of: the components of the medium are mixed.
4. A method of culturing embryonic stem cells, comprising the step of inoculating the embryonic stem cells into an embryonic stem cell culture medium, which is the embryonic stem cell culture medium of any one of claims 1 to 2;
the embryonic stem cells are huES H9 cells.
5. The method according to claim 4, wherein the embryonic stem cells are embryonic stem cells cultured to 25 th to 35 th passages.
6. The method of claim 4, further comprising: and adopting a subculture reagent for subculture in the culture process.
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